Malaria is a parasitic infection disease which is prevalent in tropical and subtropical zones. Based on statistics from the World Health Organization (WHO), 40% or more of the worldwide population lives in malaria-prevalent regions, 3 to 5 hundred million people per year are infected, and 1.5 to 3 million cases are assumed to result in death.
Malaria is mediated by a mosquito called Anopheles. When an Anopheles mosquito bites human skin, a parasite is injected into human blood together with the saliva of the Anopheles mosquito. The parasite travels into a hepatocyte, where it proliferates, and then is released again into the blood. At this time, the morphology of the parasite is referred to as a merozoite, which invades an erythrocyte soon after being released into the blood and then grows while changing its morphology. This change in the morphology is referred to as a life cycle, each stage of which is referred to as a ring form, tropozoite or schizonte. A parasite which has grown up to the stage of the schizonte destroys an erythrocyte, and then is released as a merozoite again into the blood. The released merozoite invades an erythrocyte, and repeats the life cycle again, thereby proliferating repeatedly. The malaria parasite proliferates by repeating this cycle, while destroying erythrocytes in the blood continuously. The merozoite is differentiated partly into a morphology referred to as a gametocyte without infecting the erythrocyte. The gametocyte serves as a further source of infection via the bloodsucking of an Anopheles mosquito.
The measurement method typically employed in diagnosing malaria is a method in which a blood smear preparation is stained, for example by a Giemsa staining method or a nucleic acid-staining fluorescent dye method, and then examined visually by microscopic observation using an optical microscope or a fluorescent microscope.
However, this method requires a complicated and time-consuming process involving smear preparation, fixation and staining. In addition, it requires a well-trained operator for ensuring correct detection and identification since detection and identification of the malaria parasite are performed visually. Moreover, it requires a long observation period and the observation of a large number of erythrocytes in order to ensure highly sensitive detection since a specimen to be tested from a malaria patient may have a low % infection. Accordingly, the results of the measurement may frequently be inaccurate. Moreover, it is difficult to handle a large number of specimens due to the time-consuming pretreatment and measurement stages.
Recently, methods that do not involve microscopic examination were developed, for example DNA probe method, indirect fluorescent antibody method, and indirect erythrocyte aggregation method. However, all of these methods also have disadvantages, such as complicated operation, time-consuming measurement periods, costly procedures, and the like.
In an effort to overcome these disadvantages, a method for measuring malaria parasite using flow cytometry has been developed. In this method, malaria parasite is stained with a nucleic acid-staining dye and then measured by a flow cytometer. This method mostly involves automatic measurement and involves extremely low error as compared to microscopic examination. It is also rapid and convenient since measurement times are on the order of tens of seconds to several minutes and a large number of specimens may be handled at once (for example, see J. M. Whaum et al., Cytometry, 1983, 4, 117; J. W. Jacobberger, Cytometty, 1984, 5, 589; J. D. Hare, J. Histochem. Cytochem., 1986, 34, (2) 215; and A. E. Bianco et al., Experi. Parasitol., 1986, 62, 275).
Nevertheless, the position corresponding to the appearance of malaria-infected erythrocyte may sometimes overlap with reticulocyte on the scattergram, thereby complicating the measurement of malaria-infected erythrocyte, which leads to measurement error as well as to a reduction in measurement sensitivity.
In order to solve this problem, a method was proposed in which erythrocyte is allowed to undergo hemolysis using hemolytic agent containing formaldehyde and ethylene glycol to release the malaria parasite which is then stained and measured by a flow cytometer (for example, see: P. H. Vienen et al., Cytometry, 1993, 14, 276). Alternatively, an erythrocyte is separated exclusively by centrifugation and then allowed to undergo hemolysis chemically or ultrasonically prior to counting (see: GB-A 2270752).
However, even with these methods, there are disadvantages such as complicated operation procedures (e.g., centrifugation and the like) as well as the long times required to perform the operations.
In another proposed method, erythrocyte is allowed to undergo hemolysis using a surfactant, and a blood sample containing a fluorescent-stained malaria parasite is measured using a flow cytometer to obtain forward scattering light intensity and lateral fluorescent light intensity from which a scattergram is produced and used for distinguishing and detecting various blood cells, debris (e.g., pieces of hemolytically broken erythrocyte), and malaria parasite (see: A. Saito-Ito et al., Parasitol. Int., 2001, 50(4), 249; and Japanese Unexamined Patent Publication No. 11-75892). By this method, cultured malaria can be detected at a higher sensitivity on the basis of each stage of the life cycle.